It has been found that active transport function can be restored by trypsin treatment to cells inhibited by colicin E1 if the cells are incubated at 15 degrees C. This experiment implies that the colicin molecule is exposed at the outside of the outer membrane at the time it inhibits active transport at the inner membrane. It is proposed to examine the length of the colicin molecule exposed at the outside of the outer membrane at the time of reversal of inhibitory effects by trypsin in order to define this stage in the process of penetration of the cell envelope by trypsin. Colicin El has been incorporated into membrane vesicle made of the single phospholipid dimpyristoyl phosphatidyl choline. Colicin E1 shows appreciable conductance to salts at temperatures well below the phase transition of this lipid, thus providing substantial support for a channel function of this colicin. Further studies will be made of the ion and solute selectivity of the colicin channel in this chemically well defined membrane system, as well as of the colicin conformational changes that accompany its insertion into the membrane. The orientation of colicin in this vesicle will be studied in order to define the membrane active segement. The latter studies will be complemented by using a small 20 k dalton tryptic fragment of colicin E1 which is active in the synthetic lipid vesicles.